Polymeric tandem dyes with linker groups

ABSTRACT

Compounds useful as fluorescent or colored dyes are disclosed. In some embodiments, the compounds have the following structure (I):or a stereoisomer, tautomer or salt thereof, wherein R1, R2, R3, R4, R5, L1, L2, L3, L4, M1, M2, m, and n are as defined herein. Methods associated with preparation and use of such compounds is also provided.

BACKGROUND Field

The present disclosure is generally directed to dimeric and polymericchromophore compounds (e.g., polymer compounds comprising fluorescentdye moieties) having spacing groups, and methods for their preparationand use in various analytical methods.

Description of the Related Art

Fluorescent and/or colored dyes are known to be particularly suitablefor applications in which a highly sensitive detection reagent isdesirable. Dyes that are able to preferentially label a specificingredient or component in a sample enable the researcher to determinethe presence, quantity and/or location of that specific ingredient orcomponent. In addition, specific systems can be monitored with respectto their spatial and temporal distribution in diverse environments.

Fluorescence and colorimetric methods are extremely widespread inchemistry and biology. These methods give useful information on thepresence, structure, distance, orientation, complexation and/or locationfor biomolecules. In addition, time-resolved methods are increasinglyused in measurements of dynamics and kinetics. As a result, manystrategies for fluorescence or color labeling of biomolecules, such asnucleic acids and protein, have been developed. Since analysis ofbiomolecules typically occurs in an aqueous environment, the focus hasbeen on development and use of dyes compatible with aqueous systems thatcan elucidate desired spatial information and biomolecule interactions.

Accordingly, techniques involving resonance energy transfer have beendeveloped to reveal such structural information. Specifically, Försterresonance energy transfer (“FRET”—sometimes also used interchangeablywith fluorescence resonance energy transfer) techniques produceinformation that reliably measures change in biomolecular distances andinteractions. Resonance energy transfer techniques are relatively cheapand measurements can be obtained rapidly; however, FRET suffers fromseveral limitations related to the orientation and positioning ofchromophores as well as energy transfer masking due to free fluorophoresand undesirable pH sensitivity. Additionally, known FRET reagents (e.g.,modified phycoerythrin or Brilliant Violet™) suffer from a lack ofphoto-stability, have undesirable physical characteristics (e.g., size,cell permeability), and have high batch to batch variation, whichrequires instrument recalibration.

There is a need in the art for water soluble dyes, especially resonanceenergy transfer dyes, having an increased molar brightness and/orincreased FRET emission signal. Ideally, such dyes and biomarkers shouldbe intensely colored or fluorescent and should be available in a varietyof colors and fluorescent wavelengths. The present disclosure fulfillsthis need and provides further related advantages.

BRIEF SUMMARY

In brief, embodiments of the present disclosure are generally directedto compounds useful as water soluble, fluorescent and/or colored dyesand/or probes that enable visual detection of analyte molecules, such asbiomolecules, as well as reagents for their preparation. In particular,in some embodiments, the compounds of this disclosure are useful becausethey enable FRET fluorescence emission associated with the same. Methodsfor visually detecting analyte molecules using the dyes are alsodescribed.

Embodiments of the presently disclosed dyes include two or morefluorescent and/or colored moieties (i.e., chromophores) covalentlylinked by a linker (e.g., “L⁴”). In contrast to previous reports ofprotein-based, dimeric, and/or polymeric dyes, the present dyes aresignificantly brighter, enable FRET absorbance and emission as a resultof intramolecular interactions, and are robustly reproducible usingfacile methods known in the art (i.e., automated DNA synthesis methods).

The water soluble, fluorescent or colored dyes of embodiments of thedisclosure are intensely colored and/or fluorescent, enable FRETprocesses (e.g., absorbance, emission, Stokes shifts), and can bereadily observed by visual inspection or other means. In someembodiments the compounds may be observed without prior illumination orchemical or enzymatic activation. By appropriate selection of the dye,as described herein, visually detectable analyte molecules of a varietyof colors may be obtained as well as valuable spatial information abouttarget molecules.

In one embodiment, compounds having the following structure (I) areprovided:

or a stereoisomer, tautomer or salt thereof, wherein R¹, R²,R³, R⁴, R⁵,L¹, L², L³, L₄, M¹, M², m, and n are as defined herein.

Another embodiment provides a compound having the following structure(II):

or a stereoisomer, salt or tautomer thereof, wherein R¹, R², R³, R⁴, R⁵,L¹, L², L³, L⁴, L⁵, L⁶, L^(7a), L^(7b), M¹, M³, q, w, m, and n are asdefined herein are as defined herein.

Another embodiment provides a compound having the following structure(III):

or a stereoisomer, salt, or tautomer thereof, wherein R¹, R², R₃, R⁴,R⁵, L¹, L², L³, L⁴, L⁸, M¹, M², m, and n are as define herein.

One embodiment provides a compound having the following structure (IV):

or a stereoisomer, salt or tautomer thereof, wherein R¹, R², R³, R⁴, R⁵,L¹, L², L³, L⁴, L⁵, L⁶, L^(7a), L^(7b), L⁸, M¹, M², m, and n are asdefined herein.

Yet another embodiment provides a polymer compound comprising anacceptor chromophore having an acceptor transition dipole moment andbeing covalently linked to a polymer backbone and a donor chromophorehaving a donor transition dipole moment and being covalently linked tothe polymer backbone wherein the acceptor chromophore and donorchromophore have a J-value greater than about 1×10¹⁰ and the polymercompound adopts a confirmation in solution at physiological conditionswherein the effective distance between the acceptor chromophore and thedonor chromophore is less than about 50.0 nm and the acceptor transitiondipole and the donor transition dipole are substantially parallel orsubstantially antiparallel.

The foregoing embodiments describe compounds that find utility in anumber of applications, including use as FRET, fluorescent, and/orcolored dyes in various analytical methods.

Accordingly, another embodiment provides a method for staining a sample,the method comprising adding to said sample one of the foregoingcompounds in an amount sufficient to produce an optical response whensaid sample is illuminated at an appropriate wavelength.

In still other embodiments, the present disclosure provides a method forvisually detecting an analyte molecule, comprising:

(a) providing one of the foregoing compounds (e.g., a compound ofstructure (I), (II), (III), or (IV)); and

(b) detecting the compound by its visible properties.

Other disclosed methods include a method for visually detecting abiomolecule, the method comprising:

(a) ad-mixing one of the foregoing compounds (e.g., a compound ofstructure (I), (II), (III), or (IV)) with one or more biomolecules; and

(b) detecting the compound by its visible properties.

Other embodiments are directed to a composition comprising at least oneof the foregoing compounds (e.g., a compound of structure (I), (II),(III), or (IV)) and one or more biomolecules. Use of such compositionsin analytical methods for detection of the one or more analyte (e.g.,biomolecules) is also provided.

These and other aspects of the disclosure will be apparent uponreference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are enlarged andpositioned to improve figure legibility. Further, the particular shapesof the elements as drawn are not intended to convey any informationregarding the actual shape of the particular elements, and have beensolely selected for ease of recognition in the figures.

FIG. 1 shows an exemplary donor emission spectrum and acceptorexcitation (or absorbance) spectrum and the overlap thereof used todetermine a J-value.

FIG. 2 illustrates the fluorescence emission spectra of Compound I-1using Cy3 as the donor chromophore and AF680 as the acceptor. Excitationat 488 nm.

FIG. 3 depicts the fluorescence emission spectra of Compound I-2 usingAF555 as the donor chromophore and AF680 as the acceptor. Excitation at488 nm.

FIG. 4 shows the fluorescence emission spectra of Compound I-3 using Cy3as the donor chromophore and AF647 as the acceptor. Excitation at 488nm.

FIG. 5 shows the fluorescence emission spectra of Compound I-4 usingAF555 as the donor chromophore and AF647 as the acceptor. Excitation at488 nm.

FIG. 6 depicts the fluorescence emission spectra of Compound I-5 usingCy3 as the donor chromophore and AF594 as the acceptor. Excitation at488 nm.

FIG. 7 depicts the fluorescence emission spectra of Compound I-6 usingAF555 as the donor chromophore and AF594 as the acceptor. Excitation at488 nm.

FIG. 8 displays the fluorescence emission spectra of Compound I-7 usingCy3 as the donor chromophore and AF700 as the acceptor. Excitation at488 nm.

FIG. 9 depicts the fluorescence emission spectra of Compound I-8 usingAF555 as the donor chromophore and AF700 as the acceptor. Excitation at488 nm.

FIG. 10 shows a positional overlay of the fluorescence emission spectrafor Compounds I-1, I-3, I-5 and I-7.

FIG. 11 illustrates an overlay of the emission spectra from compoundscontaining dual fluorescein moieties excited at 350 nm. Compound I-10shows increased fluorescence (relative to Compound A) as a result ofexcitation via FRET from the donor AF350 moiety.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

The term “about” has the meaning reasonably ascribed to it by a personof ordinary skill in the art when used in conjunction with a statednumerical value or range, i.e., denoting somewhat more or somewhat lessthan the stated value or range, to within a range of ±20% of the statedvalue; ±19% of the stated value; ±18% of the stated value; ±17% of thestated value; ±16% of the stated value; ±15% of the stated value; ±14%of the stated value; ±13% of the stated value; ±12% of the stated value;±11% of the stated value; ±10% of the stated value; ±9% of the statedvalue; ±8% of the stated value; ±7% of the stated value; ±6% of thestated value; ±5% of the stated value; ±4% of the stated value; ±3% ofthe stated value; ±2% of the stated value; or ±1% of the stated value.

“Amino” refers to the —NH₂ group.

“Carboxy” refers to the —CO₂H group.

“Cyano” refers to the —CN group.

“Formyl” refers to the —C(═O)H group.

“Hydroxy” or “hydroxyl” refers to the —OH group.

“Imino” refers to the ═NH group.

“Nitro” refers to the —NO2 group.

“Oxo” refers to the ═O group.

“Sulfhydryl,” “thiol,” or “thio” refers to the —SH group.

“Thioxo” refers to the =S group.

“Alkyl” refers to a straight or branched hydrocarbon chain groupconsisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to twelve carbon atoms (C₁-C₁₂ alkyl), oneto eight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, and the like. Unless stated otherwise specifically in thespecification, alkyl groups are optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, containing no unsaturation,and having from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single bond and to the radicalgroup through a single bond. The points of attachment of the alkylenechain to the rest of the molecule and to the radical group can bethrough one carbon or any two carbons within the chain. Unless statedotherwise specifically in the specification, alkylene is optionallysubstituted.

“Alkenylene” or “alkenylene chain” refers to a straight or brancheddivalent hydrocarbon chain linking the rest of the molecule to a radicalgroup, consisting solely of carbon and hydrogen, containing at least onecarbon-carbon double bond and having from two to twelve carbon atoms,e.g., ethenylene, propenylene, n-butenylene, and the like. Thealkenylene chain is attached to the rest of the molecule through asingle bond and to the radical group through a double bond or a singlebond. The points of attachment of the alkenylene chain to the rest ofthe molecule and to the radical group can be through one carbon or anytwo carbons within the chain. Unless stated otherwise specifically inthe specification, alkenylene is optionally substituted.

“Alkynylene” or “alkynylene chain” refers to a straight or brancheddivalent hydrocarbon chain linking the rest of the molecule to a radicalgroup, consisting solely of carbon and hydrogen, containing at least onecarbon-carbon triple bond and having from two to twelve carbon atoms,e.g., ethenylene, propenylene, n-butenylene, and the like. Thealkynylene chain is attached to the rest of the molecule through asingle bond and to the radical group through a double bond or a singlebond. The points of attachment of the alkynylene chain to the rest ofthe molecule and to the radical group can be through one carbon or anytwo carbons within the chain. Unless stated otherwise specifically inthe specification, alkynylene is optionally substituted. “Alkylether”refers to any alkyl group as defined above, wherein at least onecarbon-carbon bond is replaced with a carbon-oxygen bond. Thecarbon-oxygen bond may be on the terminal end (as in an alkoxy group) orthe carbon oxygen bond may be internal (i.e., C—O—C). Alkylethersinclude at least one carbon oxygen bond, but may include more than one.For example, polyethylene glycol (PEG) is included within the meaning ofalkylether. Unless stated otherwise specifically in the specification,an alkylether group is optionally substituted. For example, in someembodiments an alkylether is substituted with an alcohol or—OP(═R_(a))(R_(b))R_(c), wherein each of R_(a), R_(b) and R_(c) is asdefined for compounds of structure (I).

“Alkoxy” refers to a group of the formula —OR_(a) where R_(a) is analkyl group as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup is optionally substituted.

“Heteroalkylene” refers to an alkylene group, as defined above,comprising at least one heteroatom (e.g., Si, N, O, P, or S) within thealkylene chain or at a terminus of the alkylene chain. In someembodiments, the heteroatom is within the alkylene chain (i.e., theheteroalkylene comprises at least one carbon-[heteroatom]-carbon bond,where x is 1, 2, or 3). In other embodiments, the heteroatom is at aterminus of the alkylene and thus serves to join the alkylene to theremainder of the molecule (e.g., M_(a)-H-A-M_(b), where M_(a) and M_(b)are each a separate portion of the molecule, H is a heteroatom, and A isan alkylene). Unless stated otherwise specifically in the specification,a heteroalkylene group is optionally substituted. Exemplaryheteroalkylene groups include ethylene oxide (e.g., polyethylene oxide)and the “C linker,” “HEG linker,” and “PEG 1K linker” linking groupsillustrated below:

Multimers of the above C linker, HEG linker, and/or PEG 1K linker areincluded in various embodiments of heteroalkylene linkers. In someembodiments of the PEG 1K linker, n ranges from 19-25, for example n is19, 20, 21, 22, 23, 24, or 25. Multimers may comprise, for example, thefollowing structure:

wherein x is 0 or an integer greater than 0, for example, x ranges from0-100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

“Heteroalkenylene” is a heteroalkylene, as defined above, comprising atleast one carbon-carbon double bond. Unless stated otherwisespecifically in the specification, a heteroalkenylene group isoptionally substituted.

“Heteroalkynylene” is a heteroalkylene comprising at least onecarbon-carbon triple bond. Unless stated otherwise specifically in thespecification, a heteroalkynylene group is optionally substituted.

“Heteroatomic” in reference to a “heteroatomic linker” refers to alinker group consisting of one or more heteroatom. Exemplaryheteroatomic linkers include single atoms selected from the groupconsisting of O, N, P, and S, and multiple heteroatoms for example alinker having the formula —P(O⁻)(═O)O— or —OP(O⁻)(═O)O— and multimersand combinations thereof.

“Phosphate” refers to the —OP(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c); and R_(b) is OH, O⁻, OR_(c), a thiophosphate group ora further phosphate group, wherein R_(c) is a counter ion (e.g., Na+ andthe like).

“Phosphoalkyl” refers to the —OP(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c); and R_(b) is —Oalkyl, wherein R_(c) is a counter ion(e.g., Na⁺ and the like). Unless stated otherwise specifically in thespecification, a phosphoalkyl group is optionally substituted. Forexample, in certain embodiments, the —Oalkyl moiety in a phosphoalkylgroup is optionally substituted with one or more of hydroxyl, amino,sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether.

“Phosphoalkylether” refers to the —OP(═O)(R_(a))R_(b) group, whereinR_(a) is OH, O⁻ or OR_(c); and R_(b) is —Oalkylether, wherein R_(c) is acounter ion (e.g., Na⁺ and the like). Unless stated otherwisespecifically in the specification, a phosphoalkylether group isoptionally substituted. For example, in certain embodiments, the-Oalkylether moiety in a phosphoalkylether group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether. “Thiophosphate” refers to the—OP(═R_(a))(R_(b))R_(c) group, wherein R_(a) is O or S, R_(b) is OH, O⁻,S³¹, OR_(d) or SR_(d); and R_(c) is OH, SH, O⁻, S⁻, S⁻, OR_(d), SR_(d),a phosphate group or a further thiophosphate group, wherein R_(d) is acounter ion (e.g., Na⁺ and the like) and provided that: i) R_(a) is S;ii) R_(b) is S⁻ or SR_(d); iii) R_(c) is SH, S⁻ or SR_(d); or iv) acombination of i), ii) and/or iii).

“Thiophosphoalkyl” refers to the —OP(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is—Oalkyl, wherein R_(d) is a counter ion (e.g., Na+ and the like) andprovided that: i) R_(a) is S; ii) R_(b) is S⁻ or SR_(d); or iii)R_(a) isS and R_(b) is S⁻ or SR_(a). Unless stated otherwise specifically in thespecification, a thiophosphoalkyl group is optionally substituted. Forexample, in certain embodiments, the —Oalkyl moiety in athiophosphoalkyl group is optionally substituted with one or more ofhydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether.

“Thiophosphoalkylether” refers to the —OP(═R_(a))(R_(b))(R_(c) group,wherein R_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); andR_(c) is —Oalkylether, wherein R_(d) is a counter ion (e.g., Na⁺ and thelike) and provided that: i) R_(a) is S; ii) R_(b) is S⁻ or SR_(d); oriii)R_(a) is S and R_(b) is S⁻ or SR_(d). Unless stated otherwisespecifically in the specification, a thiophosphoalkylether group isoptionally substituted. For example, in certain embodiments, the—Oalkylether moiety in a thiophosphoalkyl group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether.

“Carbocyclic” refers to a stable 3- to 18-membered aromatic ornon-aromatic ring comprising 3 to 18 carbon atoms. Unless statedotherwise specifically in the specification, a carbocyclic ring may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems, and may be partially or fullysaturated. Non-aromatic carbocyclyl radicals include cycloalkyl, whilearomatic carbocyclyl radicals include aryl. Unless stated otherwisespecifically in the specification, a carbocyclic group is optionallysubstituted.

“Cycloalkyl” refers to a stable non-aromatic monocyclic or polycycliccarbocyclic ring, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic cyclocalkylsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Polycyclic cycloalkyls include, forexample, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless statedotherwise specifically in the specification, a cycloalkyl group isoptionally substituted.

“Aryl” refers to a ring system comprising at least one carbocyclicaromatic ring. In some embodiments, an aryl comprises from 6 to 18carbon atoms. The aryl ring may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryls include, but are not limited to, aryls derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene,indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene,and triphenylene. Unless stated otherwise specifically in thespecification, an aryl group is optionally substituted.

“Heterocyclic” refers to a stable 3- to 18-membered aromatic ornon-aromatic ring comprising one to twelve carbon atoms and from one tosix heteroatoms selected from the group consisting of nitrogen, oxygenand sulfur. Unless stated otherwise specifically in the specification,the heterocyclic ring may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclicring may be optionally oxidized; the nitrogen atom may be optionallyquaternized; and the heterocyclic ring may be partially or fullysaturated. Examples of aromatic heterocyclic rings are listed below inthe definition of heteroaryls (i.e., heteroaryl being a subset ofheterocyclic). Examples of non-aromatic heterocyclic rings include, butare not limited to, dioxolanyl, thienyl[1,3]dithianyl,decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl,isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl,piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl,pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl,trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl,thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.Unless stated otherwise specifically in the specification, aheterocyclic group is optionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system comprising one tothirteen carbon atoms, one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur, and at least one aromaticring. For purposes of certain embodiments of this disclosure, theheteroaryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzthiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,benzoxazolinonyl, benzimidazolthionyl, carbazolyl, cinnolinyl,dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl,imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl,isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl,oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl,1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl,1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl,phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyridinonyl, pyrazinyl, pyrimidinyl, pryrimidinonyl,pyridazinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl,quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl,thieno[3,2-d]pyrimidin-4-onyl, thieno[2,3-d]pyrimidin-4-onyl, triazolyl,tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless statedotherwise specifically in the specification, a heteroaryl group isoptionally substituted.

The suffix “-ene” refers to a particular structural feature (e.g.,alkyl, aryl, heteroalkyl) attached to the rest of the molecule through asingle bond and to the radical group through a single bond. In otherwords, the suffix “-ene” refers to a particular structural featurehaving the description given herein which is a linker between themolecule and a radical group. The points of attachment of the “-ene”chain to the rest of the molecule and to the radical group can bethrough one atom of or any two atoms within the chain. For example, analkyleneheteroalkylene refers to a linker comprising an alkylene portionand a heteroalkylene portion.

“Fused” refers to a ring system comprising at least two rings, whereinthe two rings share at least one common ring atom, for example twocommon ring atoms. When the fused ring is a heterocyclyl ring or aheteroaryl ring, the common ring atom(s) may be carbon or nitrogen.Fused rings include bicyclic, tricyclic, tertracyclic, and the like.

“Conjugation” refers to the overlap of one p-orbital with anotherp-orbital across an intervening sigma bond. Conjugation may occur incyclic or acyclic compounds. A “degree of conjugation” refers to theoverlap of at least one p-orbital with another p-orbital across anintervening sigma bond. For example, 1, 3-butadine has one degree ofconjugation, while benzene and other aromatic compounds typically havemultiple degrees of conjugation. Fluorescent and colored compoundstypically comprise at least one degree of conjugation.

“Fluorescent” refers to a molecule which is capable of absorbing lightof a particular frequency and emitting light of a different frequency.Fluorescence is well-known to those of ordinary skill in the art.

“Colored” refers to a molecule which absorbs light within the coloredspectrum (i.e., red, yellow, blue and the like).

“FRET” refers to Förster resonance energy transfer refers to a physicalinteraction whereby energy from the excitation of one moiety (e.g., afirst chromophore or “donor”) is transferred to an adjacent moiety(e.g., a second chromophore or “acceptor”). “FRET” is sometimes alsoused interchangeably with fluorescence resonance energy transfer (i.e.,when each chromophore is a fluorescent moiety). Generally, FRET requiresthat (1) the excitation or absorption spectrum of the acceptorchromophore overlaps with the emission spectrum of the donorchromophore; (2) the transition dipole moments of the acceptor and donorchromophores are substantially parallel (i.e., at about 0° or)180° ; and(3) the acceptor and donor chromophores share a spatial proximity (i.e.,close to each other). The transfer of energy from the donor to theacceptor occurs through non-radiative dipole-dipole coupling and thedistance between the donor chromophore and acceptor chromophore isgenerally much less than the wavelength(s) of light.

“Donor” or “donor chromophore” refers to a chromophore (e.g., afluorophore) that is or can be induced into an excited electronic stateand may transfer its excitation or absorbance energy to a nearbyacceptor chromophore in a non-radiative fashion through long-rangedipole-dipole interactions. Without wishing to be bound by theory, it isthought that the energy transfer occurs because the oscillating dipolesof the respective chromophores have similar resonance frequencies. Adonor and acceptor that have these similar resonance frequencies arereferred to as a “donor-acceptor pair(s),” which is used interchangeablywith “FRET moieties,” “FRET pairs,” “FRET dyes,” or similar. “Acceptor”or “acceptor chromophore” refers to a chromophore (e.g., a fluorophore)to which excitation or absorbance energy from a donor chromophore istransferred via a non-radiative transfer through long-rangedipole-dipole interaction.

“Stoke's shift” refers to a difference between positions (e.g.,wavelengths) of the band maxima of excitation or absorbance and emissionspectra of an electronic transition (e.g., from excited state tonon-excited state, or vice versa). In some embodiments, the compoundshave a Stoke's shift greater than 25 nm, greater than 30 nm, greaterthan 35 nm, greater than 40 nm, greater than 45 nm, greater than 50 nm,greater than 55 nm, greater than 60 nm, greater than 65 nm, greater than70 nm, greater than 75 nm, greater than 80 nm, greater than 85 nm,greater than 90 nm, greater than 95 nm, greater than 100 nm, greaterthan 110 nm, greater than 120 nm, greater than 130 nm, greater than 140nm, greater than 150 nm, greater than 160 nm, greater than 170 nm,greater than 180 nm, greater than 190 nm, or greater than 200 nm.

“J-value” is calculated as an integral value of spectral overlap betweenthe emission spectrum of a donor chromophore and the excitation orabsorbance spectrum of an acceptor chromophore. The emission spectrum ofthe donor chromophore is that which is generated when the donorchromophore is excited with a preferred excitation or absorbancewavelength. Preferred excitation or absorbance wavelengths for donorchromophores are at or near their respective excitation or absorbancemaximum well known to a person of ordinary skill in the art (e.g.,Pacific Blue has an excitation or absorbance maximum at about 401 nm,FITC has an excitation or absorbance maximum at about 495 nm). Anillustrative example of a “J-value” as used herein is shown in FIG. 1 .

A “linker” refers to a contiguous chain of at least one atom, such ascarbon, oxygen, nitrogen, sulfur, phosphorous and combinations thereof,which connects a portion of a molecule to another portion of the samemolecule or to a different molecule, moiety or solid support (e.g.,microparticle). Linkers may connect the molecule via a covalent bond orother means, such as ionic or hydrogen bond interactions.

“Physiological conditions” refers to a solution or medium having atemperature ranging from about 20 to 40° C., an atmospheric pressure ofabout 1 atm (101 kPa or 14.7 psi), a pH of about 6 to 8, a glucoseconcentration of about 1 to 20 mM, atmospheric oxygen concentration,and/or earth gravity. “Physiological conditions” includes a solution ormedium having a subset of these properties (e.g., having a temperatureranging from 20 to 40° C. and a pH of about 6 to 8). Such conditions mayalso include buffer components or systems including, but not limited tophosphate, bicarbonate, hemoglobin and/or protein.

The term “biomolecule” refers to any of a variety of biologicalmaterials, including nucleic acids, carbohydrates, amino acids,polypeptides, glycoproteins, hormones, aptamers and mixtures thereof.

More specifically, the term is intended to include, without limitation,RNA,

DNA, oligonucleotides, modified or derivatized nucleotides, enzymes,receptors, prions, receptor ligands (including hormones), antibodies,antigens, and toxins, as well as bacteria, viruses, blood cells, andtissue cells. The visually detectable biomolecules of the disclosure(e.g., compounds of structures (I), (II), (III), or (IV) having abiomolecule linked thereto) are prepared, as further described herein,by contacting a biomolecule with a compound having a reactive group thatenables attachment of the biomolecule to the compound via any availableatom or functional group, such as an amino, hydroxy, carboxyl, orsulfhydryl group on the biomolecule.

A “reactive group” is a moiety capable of reacting with a secondreactive group (e.g., a “complementary reactive group”) to form one ormore covalent bonds, for example by a displacement, oxidation,reduction, addition or cycloaddition reaction. Exemplary reactive groupsare provided in Table 1, and include for example, nucleophiles,electrophiles, dienes, dienophiles, aldehyde, oxime, hydrazone, alkyne,amine, azide, acylazide, acylhalide, nitrile, nitrone, sulfhydryl,disulfide, sulfonyl halide, isothiocyanate, imidoester, activated ester,ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide,epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, thiiraneand the like.

“Bio-conjugation” or “bio-conjugate” and related variations refer to achemical reaction strategy for forming a stable covalent bond betweentwo molecules. The term “bio-conjugation” is generally used when one ofthe molecules is a biomolecule (e.g., an antibody), but can be used todescribe forming a covalent bond with a non-biomolecule (e.g., apolymeric resin). The product or compound resulting from such a reactionstrategy is a “conjugate,” “bio-conjugate” or a grammatical equivalent.

The terms “visible” and “visually detectable” are used herein to referto substances that are observable by visual inspection, without priorillumination, or chemical or enzymatic activation. Such visuallydetectable substances absorb and emit light in a region of the spectrumranging from about 300 to about 900 nm. Preferably, such substances areintensely colored, preferably having a molar extinction coefficient ofat least about 40,000, more preferably at least about 50,000, still morepreferably at least about 60,000, yet still more preferably at leastabout 70,000, and most preferably at least about 80,000 M⁻¹cm⁻¹. Thecompounds of the disclosure may be detected by observation with thenaked eye, or with the aid of an optically based detection device,including, without limitation, absorption spectrophotometers,transmission light microscopes, digital cameras and scanners. Visuallydetectable substances are not limited to those which emit and/or absorblight in the visible spectrum. Substances which emit and/or absorb lightin the ultraviolet (UV) region (about 10 nm to about 400 nm), infrared(IR) region (about 700 nm to about 1 mm), and substances emitting and/orabsorbing in other regions of the electromagnetic spectrum are alsoincluded with the scope of “visually detectable” substances.

For purposes of embodiments of the disclosure, the term “photostablevisible dye” refers to a chemical moiety that is visually detectable, asdefined hereinabove, and is not significantly altered or decomposed uponexposure to light. Preferably, the photostable visible dye does notexhibit significant bleaching or decomposition after being exposed tolight for at least one hour. More preferably, the visible dye is stableafter exposure to light for at least 12 hours, still more preferably atleast 24 hours, still yet more preferably at least one week, and mostpreferably at least one month. Non-limiting examples of photostablevisible dyes suitable for use in the compounds and methods of thedisclosure include azo dyes, thioindigo dyes, quinacridone pigments,dioxazine, phthalocyanine, perinone, diketopyrrolopyrrole,quinophthalone, and truarycarbonium.

As used herein, the term “perylene derivative” is intended to includeany substituted perylene that is visually detectable. However, the termis not intended to include perylene itself. The terms “anthracenederivative”, “naphthalene derivative”, and “pyrene derivative” are usedanalogously. In some preferred embodiments, a derivative (e.g.,perylene, pyrene, anthracene or naphthalene derivative) is an imide,bisimide or hydrazamimide derivative of perylene, anthracene,naphthalene, or pyrene.

The polymer compounds of various embodiments of the disclosure areuseful for a wide variety of analytical applications, such asbiochemical and biomedical applications, in which there is a need todetermine the presence, location, spatial interaction or quantity of aparticular analyte (e.g., biomolecule). In another aspect, therefore,the disclosure provides a method for visually detecting a biomolecule,comprising: (a) providing a biological system with a visually detectablebiomolecule comprising the compound of the embodiments disclosed herein(e.g., compounds of structures (I), (II), (III), or (IV)) linked to abiomolecule; and (b) detecting the biomolecule by its visibleproperties. For purposes of the disclosure, the phrase “detecting thebiomolecule by its visible properties” means that the biomolecule,without illumination or chemical or enzymatic activation, is observedwith the naked eye, or with the aid of a optically based detectiondevice, including, without limitation, absorption spectrophotometers,transmission light microscopes, digital cameras and scanners. Adensitometer may be used to quantify the amount of visually detectablebiomolecule present. For example, the relative quantity of thebiomolecule in two samples can be determined by measuring relativeoptical density. If the stoichiometry of dye molecules per biomoleculeis known, and the extinction coefficient of the dye molecule is known,then the absolute concentration of the biomolecule can also bedetermined from a measurement of optical density. As used herein, theterm “biological system” is used to refer to any solution or mixturecomprising one or more biomolecules in addition to the visuallydetectable biomolecule. Non-limiting examples of such biological systemsinclude cells, cell extracts, tissue samples, electrophoretic gels,assay mixtures, and hybridization reaction mixtures.

“Solid support” or “solid support residue” refers to any solid substrateknown in the art for solid-phase support of molecules, for example a“microparticle” refers to any of a number of small particles useful forattachment to compounds of the disclosure, including, but not limitedto, glass beads, magnetic beads, polymeric beads, non-polymeric beads,and the like. In certain embodiments, a microparticle comprisespolystyrene beads.

“Base pairing moiety” refers to a heterocyclic moiety capable ofhybridizing with a complementary heterocyclic moiety via hydrogen bonds(e.g., Watson-Crick base pairing). Base pairing moieties include naturaland unnatural bases.

Non-limiting examples of base pairing moieties are RNA and DNA basessuch adenosine, guanosine, thymidine, cytosine and uridine and analoguesthereof.

Embodiments of this disclosure are also meant to encompass all compoundsof structure (I) or (II) being isotopically-labelled by having one ormore atoms replaced by an atom having a different atomic mass or massnumber. Examples of isotopes that can be incorporated into the disclosedcompounds include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C,¹⁴C, ¹³ N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I,respectively. Isotopically-labeled compounds of structure (I) or (II)can generally be prepared by conventional techniques known to thoseskilled in the art or by processes analogous to those described belowand in the following Examples using an appropriate isotopically-labeledreagent in place of the non-labeled reagent previously employed.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Optional” or “optionally” means that the subsequently described eventor circumstances may or may not occur; such a description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, “optionally substituted alkyl” means that thealkyl group may or may not be substituted and that the descriptionincludes both substituted alkyl groups and alkyl groups having nosubstitution.

“Salt” includes both acid and base addition salts.

“Acid addition salt” refers to those salts which are formed withinorganic acids such as, but not limited to, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as, but not limited to, acetic acid,2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproicacid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid,lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid,malonic acid, mandelic acid, methanesulfonic acid, mucic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid,oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamicacid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid,stearic acid, succinic acid, tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and thelike.

“Base addition salt” refers to those salts which are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Salts derived from organic basesinclude, but are not limited to, salts of primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asammonia, isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Crystallizations may produce a solvate of the compounds describedherein. Embodiments of the present disclosure include all solvates ofthe described compounds. As used herein, the term “solvate” refers to anaggregate that comprises one or more molecules of a compound of thedisclosure with one or more molecules of solvent. The solvent may bewater, in which case the solvate may be a hydrate. Alternatively, thesolvent may be an organic solvent. Thus, the compounds of the presentdisclosure may exist as a hydrate, including a monohydrate, dihydrate,hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, aswell as the corresponding solvated forms. The compounds of thedisclosure may be true solvates, while in other cases the compounds ofthe disclosure may merely retain adventitious water or another solventor be a mixture of water plus some adventitious solvent.

Embodiments of the compounds of the disclosure (e.g., compounds ofstructure I, II, III or IV), or their salts, tautomers or solvates maycontain one or more stereocenters and may thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that may be defined, interms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)-for amino acids. Embodiments of the present disclosure are meant toinclude all such possible isomers, as well as their racemic andoptically pure forms. Optically active (+) and (−), (R)- and (5)-, or(D)- and (L)- isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques, for example,chromatography and fractional crystallization. Conventional techniquesfor the preparation/isolation of individual enantiomers include chiralsynthesis from a suitable optically pure precursor or resolution of theracemate (or the racemate of a salt or derivative) using, for example,chiral high pressure liquid chromatography (HPLC). When the compoundsdescribed herein contain olefinic double bonds or other centers ofgeometric asymmetry, and unless specified otherwise, it is intended thatthe compounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present disclosure contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are non-superimposablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present disclosure includestautomers of any said compounds. Various tautomeric forms of thecompounds are easily derivable by those of ordinary skill in the art.

The chemical naming protocol and structure diagrams used herein are amodified form of the I.U.P.A.C. nomenclature system, using the ACD/NameVersion 9.07 software program and/or ChemDraw Ultra Version 11.0software naming program (CambridgeSoft). Common names familiar to one ofordinary skill in the art are also used.

As noted above, in one embodiment of the present disclosure, compoundsuseful as FRET, fluorescent, and/or colored dyes in various analyticalmethods are provided. In other embodiments, compounds useful assynthetic intermediates for preparation of compounds useful as FRET,fluorescent, and/or colored dyes are provided. In general terms,embodiments of the present disclosure are directed to dimers, trimers,and higher polymers of FRET, fluorescent, and/or colored moieties. TheFRET, fluorescent, and/or colored moieties are linked by a linkingmoiety. Without wishing to be bound by theory, it is believed the linkerhelps to maintain sufficient spatial distance/proximity between thedonor-acceptor pair(s) such that intramolecular quenching is reduced oreliminated, while maintain sufficient proximity to facilitate thenon-radiative transfer of energy.

Accordingly, in some embodiments the compounds have the followingstructure

wherein L is a linker sufficient to maintain spatial separation betweenone or more (e.g., each) M group so that intramolecular quenching isreduced or eliminated, and R¹, R², R³, L¹, L², L³ and n are as definedfor structure (I), (II), (III), or structure (IV).

In other embodiments is provided a compound having the followingstructure (I):

or a stereoisomer, salt, or tautomer thereof, wherein:

M¹ and M² are, at each occurrence, independently a chromophore, providedthat at least one of M¹ and M² is a FRET donor, and another one of M¹and M² is a corresponding FRET acceptor;

L¹ is, at each occurrence, an optional linker, provided that at leastone occurrence of L¹ is present and comprises oxygen;

L² and L³ are, at each occurrence, independently an optional alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker;

L⁴ is, at each occurrence, independently an alkylene or heteroalkylenelinker;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,heteroalkyl, —OP(═R_(a))(R_(b))R_(c), Q, or a protected form thereof, orL′;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(d) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

R_(a) is O or S;

R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d);

R_(c) is OH, SH, O⁻, S⁻, OR_(d), OL′, SR_(d), alkyl, alkoxy,heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether;

R_(d) is a counter ion;

Q is, at each occurrence, independently a moiety comprising a reactivegroup, or protected form thereof, capable of forming a covalent bondwith an analyte molecule, a targeting moiety, a solid support, or acomplementary reactive group Q′;

L¹ is, at each occurrence, independently a linker comprising a covalentbond to Q, a linker comprising a covalent bond to a targeting moiety, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support, a linker comprising acovalent bond to a solid support residue, a linker comprising a covalentbond to a nucleoside or a linker comprising a covalent bond to a furthercompound of structure (I);

m is, at each occurrence, independently an integer of zero or greater;and

n is an integer of one or greater.

In more specific embodiments, at least one occurrence of L⁴ isheteroalkylene when M¹ and M² are selected from fluorescein, pyrene, andperylene.

Some other embodiments provide a compound having the following structure(II):

or a stereoisomer, salt, or tautomer thereof, wherein:

M¹ and M³ are, at each occurrence, independently a chromophore, providedthat at least one of M¹ and M³ is a FRET donor, and another one of M¹and M³ is a corresponding FRET acceptor;

L¹, L^(7a), and L^(7b) are, at each occurrence, independently anoptional linker;

L², L³, L⁵ and L⁶ are, at each occurrence, independently an optionalalkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker;

L⁴ is, at each occurrence, independently an alkylene or heteroalkylenelinker;

R¹ is, at each occurrence, independently H, alkyl, or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,heteroalkyl, —OP(═R_(a))(R_(b))R_(c), Q, or a protected form thereof, orL′;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(d) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

R_(a) is O or S;

R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d);

R^(c) is OH, SH, O⁻, S⁻, OR_(d), OL′, SR^(d), alkyl, alkoxy,heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether;

R^(d) is a counter ion;

Q is, at each occurrence, independently a moiety comprising a reactivegroup, or protected form thereof, capable of forming a covalent bondwith an analyte molecule, a targeting moiety, a solid support or acomplementary reactive group Q;

L¹ is, at each occurrence, independently a linker comprising a covalentbond to Q, a linker comprising a covalent bond to a targeting moiety, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support, a linker comprising acovalent bond to a solid support residue, a linker comprising a covalentbond to a nucleoside or a linker comprising a covalent bond to a furthercompound of structure (II);

m is, at each occurrence, independently an integer of zero or greater;and

n is an integer of one or greater;

q is an integer of zero or greater, provided that q is an integer of oneor greater for at least one occurrence; and

w is an integer of zero or greater, provided that q is an integer of oneor greater for at least one occurrence.

Some other embodiments provide a compound having the following structure(III):

or a stereoisomer, salt, or tautomer thereof, wherein:

M¹ and M² are, at each occurrence, independently a chromophore, providedthat at least two occurrences of M¹ are independently FRET donors, andat least one occurrence of M² is a FRET acceptor that forms a FRET pairwith each of the at least two occurrences of M¹;

L¹ is, at each occurrence, an optional linker, provided that at leastone occurrence of L¹ is present and comprises oxygen;

L² and L³ are, at each occurrence, independently an optional alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker;

L⁴ is, at each occurrence, independently an alkylene or heteroalkylenelinker;

L⁸ is, at each occurrence, independently a rigid linker;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,heteroalkyl, —OP(═R_(a))(R_(b))R_(c), Q, or a protected form thereof, orL′;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(d) orSR_(a);

R⁵ is, at each occurrence, independently oxo, thioxo, or absent;

R_(a) is O or S;

R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d);

R_(c) OH, SH, O⁻, S⁻, OR_(d), OL′, SR_(d), alkyl, alkoxy, heteroalkyl,heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate,phosphoalkyl, thiophosphoalkyl, phosphoalkylether, orthiophosphoalkylether;

R_(d) is a counter ion;

Q is, at each occurrence, independently a moiety comprising a reactivegroup, or protected form thereof, capable of forming a covalent bondwith an analyte molecule, a targeting moiety, a solid support or acomplementary reactive group Q′;

L¹ is, at each occurrence, independently a linker comprising a covalentbond to Q, a linker comprising a covalent bond to a targeting moiety, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support, a linker comprising acovalent bond to a solid support residue, a linker comprising a covalentbond to a nucleoside or a linker comprising a covalent bond to a furthercompound of structure (III);

m is, at each occurrence, independently an integer of zero or greater;and

n is an integer of one or greater.

In more specific embodiments, at least one occurrence of L⁴ isheteroalkylene when M¹ and M² are selected from fluorescein, pyrene, andperylene.

One specific embodiment provides a compound having the followingstructure (IV):

or a stereoisomer, salt, or tautomer thereof, wherein:

M¹ and M³ are, at each occurrence, independently a chromophore, providedthat at least two occurrences of M¹ are independently FRET donors, andat least one occurrence of M³ is a FRET acceptor that forms a FRET pairwith each of the at least two occurrences of M¹;

L¹, L⁷a, and L^(7b) are, at each occurrence, independently an optionallinker;

L², L³, L⁵ and L⁶ are, at each occurrence, independently an optionalalkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker;

L⁴ is, at each occurrence, independently an alkylene or heteroalkylenelinker;

L⁸ is, at each occurrence, independently a rigid linker;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,heteroalkyl, —OP(═R_(a))(R_(b))R_(c), Q, or a protected form thereof, orL′;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(d) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

R_(a) O or S;

R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d);

R_(c) is OH, SH, O⁻, S⁻, OR_(d), OL′, SR_(d), alkyl, alkoxy,heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether;

R_(d) is a counter ion;

Q is, at each occurrence, independently a moiety comprising a reactivegroup, or protected form thereof, capable of forming a covalent bondwith an analyte molecule, a targeting moiety, a solid support or acomplementary reactive group Q′;

L¹ is, at each occurrence, independently a linker comprising a covalentbond to Q, a linker comprising a covalent bond to a targeting moiety, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support, a linker comprising acovalent bond to a solid support residue, a linker comprising a covalentbond to a nucleoside or a linker comprising a covalent bond to a furthercompound of structure (IV);

m is, at each occurrence, independently an integer of zero or greater;and

n is an integer of one or greater.

In some of the foregoing embodiment, at least one occurrence of L⁴ isheteroalkylene. In some embodiments, each occurrence of L⁴ isheteroalkylene. In more specific embodiments, the heteroalkylenecomprises alkylene oxide. In some related embodiments, theheteroalkylene comprises ethylene oxide.

In embodiments, at least one occurrence of L⁴ is alkylene. In some morespecific embodiments, at least one alkylene is ethylene. In someembodiments, the alkylene is ethylene at each occurrence.

In some more specific embodiments, the compound has the followingstructure (IA):

wherein:

z is, at each occurrence, independently an integer from 1 to 100; and

m is, at each occurrence, independently an integer from 0 to 6.

In other embodiments, the compound has the following structure (IIA):

wherein:

z is, at each occurrence, independently an integer from 1 to 100; and

m is, at each occurrence, independently an integer from 0 to 6.

In certain embodiments, the compound has the following structure

(IIIA):

wherein:

L⁴ is —(OCH₂CH₂)_(z)—;

z is, at each occurrence, independently an integer from 1 to 100; and

m is, at each occurrence, independently an integer from 0 to 6.

In certain other embodiments, the compound has the following structure

(IVA):

wherein:

L⁴ is —(OCH₂CH₂)_(z)—;

z is, at each occurrence, independently an integer from 1 to 100; and

m is, at each occurrence, independently an integer from 0 to 6.

In some embodiments, z is, at each occurrence, independently an integerfrom 1 to 30. In certain embodiments, z is, at each occurrence,independently 3 or 6. In other embodiments, m is, at each occurrence,independently an integer from 2-4. In some embodiments, m is 2 at eachoccurrence.

In some more specific embodiments, the compound has the followingstructure (IB):

wherein:

x¹, x², x³, and x⁴ are, at each occurrence, independently an integerfrom 0 to 6.

In certain embodiments, the compound has the following structure (IIB):

wherein:

x¹, x², x³, and x⁴ are, at each occurrence, independently an integerfrom 0 to 6.

In certain more specific embodiments, the compound has the followingstructure (IIIB):

wherein:

x¹, x², x³, x⁴, x⁵, and x⁶ are, at each occurrence, independently aninteger from 0 to 6.

In certain embodiments, the compound has the following structure (IVB):

wherein:

x¹, x², x³, x⁴, x⁵, and x⁶ are, at each occurrence, independently aninteger from 0 to 6.

In certain related embodiments, z is an integer from 3 to 6 at one ormore occurrences. In some specific embodiments, x¹ and x³ are both 0 ateach occurrence, and x² and x⁴ are both 1 at each occurrence. In someembodiments, x¹, x², x³ and x⁴ are all 1 at each occurrence. In someembodiments, x¹, x³, and x⁵ are all 1 at each occurrence, and x², x⁴,and x⁶ are all 0 at each occurrence. In certain embodiments, x¹, x², x³,x⁴, x⁵, and x⁶ are all 1 at each occurrence.

In one particular embodiment, L⁴ has the following structure:

wherein:

p is, at each occurrence, independently an integer from 0 to 6; and

y is, at each occurrence, independently an integer from 1 to 100.

In some embodiments, R³ comprises the following structure:

wherein:

p is, at each occurrence, independently an integer from 0 to 6; and

y is, at each occurrence, independently an integer from 1 to 100.

The various linkers and substituents (e.g., M¹, M², M³, Q, R¹, R², R³,R^(c) L¹, L², L³, L⁴, L⁵, L⁵, L⁶, L^(7a), L^(7b), R⁸) in the compoundsof structures (I), (II), (III) or ,

(IV) are optionally substituted with one more substituent. For example,in some embodiments the optional substituent is selected to optimize thewater solubility or other property of the compounds of structures (I),(II), (III), or (IV). In certain embodiments, each alkyl, alkoxy,alkylether , alkoxyalkylether, phosphoalkyl, thiophosphoalkyl,phosphoalkylether and thiophosphoalkylether in the compounds ofstructures (I), (II), (III), or (IV) are optionally substituted with onemore sub stituent selected from the group consisting of hydroxyl,alkoxy, alkylether , alkoxyalkylether, sulfhydryl, amino, alkylamino,carboxyl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether and thiophosphoalkylether.

In some embodiments, L¹ or L^(7b) is, at each occurrence, independentlyan optional alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, heteroalkynylene, or heteroatomic linker. In someembodiments, L¹ or L^(7b) is a linker comprising a functional groupcapable of formation by reaction of two complementary reactive groups(e.g., an azide and an alkyne). In some embodiments, L¹ or L^(7b) is, ateach occurrence, independently an optional alkylene, alkenylene,alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene,alkyleneheteroarylenealkylene, alkyleneheterocyclylenealkylene,alkylenecarbocyclylenealkylene, heteroalkyleneheteroarylenealkylene,heteroalkyleneheterocyclylenealkylene,heteroalkylenecarbocyclylenealkylene,heteroalkyleneheteroaryleneheteroalkylene,heteroalkyleneheterocyclyleneheteroalkylene,heteroalkylenecarbocyclyleneheteroalkylene,alkyleneheteroaryleneheteroalkylene,alkyleneheterocyclyleneheteroalkylene,alkylenecarbocyclyleneheteroalkylene, heteroarylene, heterocyclylene,carbocyclylene, alkyleneheteroarylene, alkyleneheterocyclylene,heteroarylenealkylene, alkylenecarbocyclylene, carbocyclylenealkylene,heteroalkyleneheteroarylene, heteroalkyleneheterocyclylene,heteroaryleneheteroalkylene, heteroalkylenecarbocyclylene,carbocyclyleneheteroalkylene, or heteroatomic linker. In someembodiments, L¹ or is optionally substituted.

The optional linkers L¹ and L^(7b) can be used as a point of attachmentof the M¹, M², and M³ moieties to the remainder of the compound. Forexample, in some embodiments a synthetic precursor to the compound ofstructure (I), (II), (III), or (IV) is prepared, and the M¹, M², and/orM³ moiety is attached to the synthetic precursor using any number offacile methods known in the art, for example methods referred to as“click chemistry.” For this purpose any reaction which is rapid andsubstantially irreversible can be used to attach M¹, M², or M³ or bothto the synthetic precursor to form a compound of structure (I), (II),(III), or (IV). Exemplary reactions include the copper catalyzedreaction of an azide and alkyne to form a triazole (Huisgen 1, 3-dipolarcycloaddition), reaction of a diene and dienophile (Diels-Alder),strain-promoted alkyne-nitrone cycloaddition, reaction of a strainedalkene with an azide, tetrazine or tetrazole, alkene and azide [3+2]cycloaddition, alkene and tetrazine inverse-demand Diels-Alder, alkeneand tetrazole photoreaction and various displacement reactions, such asdisplacement of a leaving group by nucleophilic attack on anelectrophilic atom. In some embodiments the reaction to form L¹ orL^(7b) may be performed in an aqueous environment.

Accordingly, in some embodiments L¹ or L^(7b) is at each occurrence alinker comprising a functional group capable of formation by reaction oftwo complementary reactive groups, for example a functional group whichis the product of one of the foregoing “click” reactions. In variousembodiments, for at least one occurrence of L¹ or L^(7b), the functionalgroup can be formed by reaction of an aldehyde, oxime, hydrazone,alkyne, amine, azide, acylazide, acylhalide, nitrile, nitrone,sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imidoester,activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide,α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotinor thiirane functional group with a complementary reactive group.

In other embodiments, for at least one occurrence of L¹ or L^(7b), thefunctional group can be formed by reaction of an alkyne and an azide.

In more embodiments, for at least one occurrence of L¹ or L^(7b), thefunctional group comprises an alkene, ester, amide, thioester,disulfide, carbocyclic, heterocyclic, or heteroaryl group. In some morespecific embodiments, for at least one occurrence of L¹ or L^(7b), L¹ orL^(7b) is a linker comprising a triazolyl functional group.

In some embodiments, at least one occurrence of L^(7b)-M³ comprises thefollowing structure:

wherein L^(a) and L^(b) are each independently optional linkers.

In certain embodiments, at least one occurrence of L¹-M¹ or L¹-M²comprises one of the following structures:

wherein L^(a) and L^(b) are each independently optional linkers. In someembodiments, at least one occurrence of L^(7b)-M³ comprises thefollowing structure:

wherein L^(a) and L^(b) are each independently optional linkers.

In certain embodiments, L^(a) or L^(b), or both, is absent. In someembodiments, L^(a) or L^(b), or both, is present. In some more specificembodiments, L^(a) and L^(b), when present, are each independentlyalkylene or heteroalkylene. In still other embodiments, L^(a) and L^(b)independently have one of the following structures:

In still other different embodiments, L¹ or L^(7b) is at eachoccurrence, independently an optional alkylene or heteroalkylene linker.

In some embodiments, at least one occurrence of L¹ has one of thefollowing structures:

wherein

a, b, and c are each independently an integer ranging from 1-6.

In some embodiments, each occurrence of L¹ has one of the followingstructures:

wherein

a, b, and c are each independently an integer ranging from 1-6.

In some embodiments, at least one occurrence of L¹ has one of thefollowing structures:

In some embodiments, each occurrence of L¹ has one of the followingstructures:

In some embodiments, L^(7a) a comprises an optionally substituted 5-7membered heteroarylene linker. In certain embodiments, L^(7a) is, ateach occurrence independently an optionally substituted 5-7 memberedheteroarylene linker. In some more specific embodiments, L^(7a) is a6-membered heteroarylene. In certain more specific embodiments, L^(7a)comprises two N atoms and two O atoms. In some embodiments, L^(7a) is,at each occurrence, substituted. In certain embodiments, L^(7a) issubstituted with at least one oxo. In some more specific embodiments,L^(7a) has one of the following structures:

In still other embodiments, R⁴ is, at each occurrence, independently OH,O⁻or OR_(d). It is understood that “OR_(d)” and “SR_(a)” are intended torefer to O⁻ and S⁻associated with a cation. For example, the disodiumsalt of a phosphate group may be represented as:

where R_(a) is sodium (Na³⁰).

In other embodiments, R⁵ is, at each occurrence, oxo.

In some different embodiments of any of the foregoing compounds, le isH.

In other various embodiments, R² and R³ are each independently OH or—OP(═R_(a))(R_(b))R_(c). In some different embodiments, R² or R³ is OHor —OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is Q or a linkercomprising a covalent bond to Q. In some embodiments, R² and R³ are eachindependently —OP(═R_(a))(R_(b))R_(c). In some specific embodiments,R_(c) is OL′. In some of those embodiments, L′ is a heteroalkylenelinker to: Q, a targeting moiety, an analyte molecule, a solid support,a solid support residue, a nucleoside or a further compound of structure(I), (II), (III), or (IV). In some embodiments, L′ comprises an alkyleneoxide or phosphodiester moiety, or combinations thereof. In certainembodiments, L′ has the following structure:

wherein:

m″ and n″ are independently an integer from 1 to 10;

R_(e) is H, an electron pair or a counter ion;

L″ is R_(e) or a direct bond or linkage to: Q, a targeting moiety, ananalyte molecule, a solid support, a solid support residue, a nucleosideor a further compound of structure (I), (II), (III), or (IV).

In still other embodiments, Q is, at each occurrence, independently amoiety comprising a reactive group capable of forming a covalent bondwith an analyte molecule or a solid support (e.g., controlled pore glassor polystyrene beads). In other embodiments, Q is, at each occurrence,independently a moiety comprising a reactive group capable of forming acovalent bond with a complementary reactive group Q′. For example, insome embodiments, Q′ is present on a further compound of structure (I),(II), (III), or (IV) (e.g., in the R² or R³ position), and Q and Q′comprise complementary reactive groups such that reaction of thecompound of structure (I), (II), (III), or (IV) and the further compoundof structure (I), (II), (III), or (IV) results in covalently bound dimerof the compound of structure (I), (II), (III), or (IV). Multimercompounds of structures (I), (II), (III), or (IV), and combinationsthereof can also be prepared in an analogous manner and are includedwithin the scope of embodiments of the disclosure.

The type of Q group and connectivity of the Q group to the remainder ofthe compound of structure (I), (II), (III), or (IV) is not particularlylimited, provided that Q comprises a moiety having appropriatereactivity for forming the desired bond.

In certain embodiments, Q is a moiety which is not susceptible tohydrolysis under aqueous conditions, but is sufficiently reactive toform a bond with a corresponding group on an analyte molecule (e.g., abiomolecule) or solid support (e.g., an amine, azide, or alkyne).

Certain embodiments of compounds of structure (I), (II), (III), and/or(IV) comprise Q groups commonly employed in the field ofbio-conjugation. For example in some embodiments, Q comprises anucleophilic reactive group, an electrophilic reactive group or acycloaddition reactive group. In some more specific embodiments, Qcomprises a sulfhydryl, disulfide, activated ester, isothiocyanate,azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide,phosphine, a-haloamide, biotin, amino, or maleimide functional group. Insome embodiments, the activated ester is an N-succinimide ester,imidoester, or polyflourophenyl ester. In other embodiments, the alkyneis an alkyl azide or acyl azide. In some embodiments, Q comprises amaleimide functional group.

Exemplary Q moieties are provided in Table I below.

TABLE 1 Exemplary Q Moieties Structure Class

Sulfhydryl

Isothiocyanate

Imidoester

Acyl Azide

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Sulfonyl halide X = halo

Maleimide

Maleimide

α-haloimide X = halo

Disulfide

Phosphine

Azide

Alkyne

Biotin

Diene

Alkene/dienophile

Alkene/dienophile EWG = eletron withdrawing group

Amino

It should be noted that in some embodiments, wherein Q is SH, the SHmoiety will tend to form disulfide bonds with another sulfhydryl groupon another compound of structure (I), (II), (III), or (IV). Accordingly,some embodiments include compounds of structure (I), (II), (III), or(IV), which are in the form of disulfide dimers, the disulfide bondbeing derived from SH Q groups.

In some other embodiments, one of R² or R³ is OH or—OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is a linkercomprising a covalent bond to an analyte molecule or a linker comprisinga covalent bond to a solid support. For example, in some embodiments theanalyte molecule is a nucleic acid or a polymer thereof or an amino acidor a polymer thereof. In other embodiments, the analyte molecule is anenzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer orprion. In still different embodiments, the solid support is a polymericbead or non-polymeric bead. In some embodiments, the targeting moiety isan antibody or cell surface receptor antagonist.

In certain specific embodiments, R² or R³ has one of the followingstructures:

In some embodiments, one of R² or R³ is OH or —OP(═R_(a))(R_(b))R_(c),and the other of R² or R³ comprises the following structure:

The value for m is another variable that can be selected based on thedesired distance between chromophores, FRET interaction, fluorescence,and/or color intensity. In some embodiments, m is, at each occurrence,independently an integer from 1 to 10. In other embodiments, m is, ateach occurrence, independently an integer from 1 to 5, for example 1, 2,3, 4 or 5.

The fluorescence intensity can also be tuned by selection of differentvalues of n. In certain embodiments, n is an integer from 1 to 100. Inother embodiments, n is an integer from 1 to 10.

M¹, M², and M³ are selected based on the desired optical properties, forexample based on a desired Stoke's shift, absorbance/emission overlap, aparticular color and/or fluorescence emission wavelength. In someembodiments, M¹, M², or M³ are the same at each occurrence; however, itis important to note that each occurrence of

M¹, M², or M³ need not be an identical M¹, M², or M³, respectively.Certain embodiments include compounds wherein M¹, M², or M³ is not thesame at each occurrence. Some embodiments include compounds wherein eachrespective M¹, M², or M³ is the same at each occurrence.

In some embodiments M¹, M², and M³ are selected to have absorbanceand/or emission characteristics for use in FRET methods. For example, insuch embodiments the different M¹, M², and M³ moieties are selected suchthat M¹ has an absorbance of radiation at one wavelength that induces anemission of radiation by M² or M³ at a different wavelength by a FRETmechanism. Exemplary M¹, M², and M³ moieties can be appropriatelyselected by one of ordinary skill in the art based on the desired enduse.

Each respective M¹, M², and M³ may be attached to the remainder of themolecule from any position (i.e., atom) on M¹, M², or M³. One of skillin the art will recognize means for attaching M¹, M², or M³ to theremainder of molecule. Exemplary methods include the “click” reactionsdescribed herein.

In some embodiments, M¹, M², or M³ are FRET, fluorescent, or coloredmoieties. Any fluorescent and/or colored moiety may be used to form aFRET donor-acceptor pair, for examples those known in the art andtypically employed in colorimetric, UV, and/or fluorescent assays may beused. In some embodiments, M¹, M², M³, or all are, at each occurrence,independently fluorescent or colored. Examples of M¹, M², or M³ moietieswhich are useful in various embodiments of the disclosure include, butare not limited to: Xanthene derivatives (e.g., fluorescein, rhodamine,Oregon green, eosin, or Texas red); Cyanine derivatives (e.g., cyanine,indocarbocyanine, oxacarbocyanine, thiacarbocyanine, or merocyanine);Squaraine derivatives and ring-substituted squaraines, including Seta,SeTau, and Square dyes; Naphthalene derivatives (e.g., dansyl and prodanderivatives); Coumarin derivatives; oxadiazole derivatives (e.g.,pyridyloxazole, nitrobenzoxadiazole, or benzoxadiazole); Anthracenederivatives (e.g., anthraquinones, including DRAQS, DRAQ7, and CyTRAKOrange); Pyrene derivatives such as cascade blue; Oxazine derivatives(e.g., Nile red, Nile blue, cresyl violet, oxazine 170); Acridinederivatives (e.g., proflavin, acridine orange, acridine yellow);Arylmethine derivatives: auramine, crystal violet, malachite green; andTetrapyrrole derivatives (e.g., porphin, phthalocyanine or bilirubin).Other exemplary M¹, M^(2,) or M³ moieties include: Cyanine dyes,xanthate dyes (e.g., Hex, Vic, Nedd, Joe or Tet); Yakima yellow; Redmondred; tamra; texas red and Alexa fluor® dyes.

In still other embodiments of any of the foregoing, M¹,M², or M³, orboth, at each occurrence, independently comprise two or more aryl orheteroaryl rings, or combinations thereof, for example three or more orfour or more aryl or heteroaryl rings, or combinations thereof, or evenfive or more aryl or heteroaryl rings, or combinations thereof. In someembodiments, M¹, M², M³, or all, at each occurrence, independentlycomprise six aryl or heteroaryl rings, or combinations thereof. Infurther embodiments, the rings are fused. For example in someembodiments, M¹, M², or M³ or both, at each occurrence, independentlycomprise two or more fused rings, three or more fused rings, four ormore fused rings, five or more fused rings, or even six or more fusedrings.

In some embodiments, M¹, M², M³, or all, are cyclic. For example, insome embodiments M¹, M², M³, or all, are carbocyclic. In otherembodiment, M¹, M², M³, or all are heterocyclic. In still otherembodiments of the foregoing, M¹, M², M³, or all, at each occurrence,independently comprise an aryl moiety. In some of these embodiments, thearyl moiety is multicyclic. In other more specific examples, the arylmoiety is a fused-multicyclic aryl moiety, for example which maycomprise at least 2, at least 3, at least 4, or even more than 4 arylrings.

In other embodiments of any of the foregoing compounds of structure (I),(II), (III), or (IV) M¹, M², M3, or all, at each occurrence,independently comprise at least one heteroatom. For example, in someembodiments, the heteroatom is nitrogen, oxygen or sulfur.

In still more embodiments of any of the foregoing, M², M3, or all, ateach occurrence, independently comprise at least one substituent. Forexample, in some embodiments the substituent is a fluoro, chloro, bromo,iodo, amino, alkylamino, arylamino, hydroxy, sulfhydryl, alkoxy,aryloxy, phenyl, aryl, methyl, ethyl, propyl, butyl, isopropyl, t-butyl,carboxy, sulfonate, amide, or formyl group.

In some embodiments, at least one combination of M¹ and M² or at leastone combination of M¹ and M³ are a FRET pair with a J-value greater thanabout 1×10¹⁰.

Compounds of the present disclosure find utility as fluorescent and/orcolored dyes with high quantum efficiencies. This is due, in part, tothe overlap of the emission spectrum of a donor moiety (e.g., M¹) withthe absorbance or excitation spectrum of an acceptor moiety (e.g., M¹,M² or M³). Accordingly, some embodiments provide a FRET pair having aJ-value greater than about 1×10¹¹. In more specific embodiments, theFRET pair has a J-value greater than about 1×10¹². In certainembodiments, the FRET pair has a J-value greater than about 1.2×10¹². Insome embodiments, the FRET pair has a J-value greater than about1.5×10¹².

In some more specific embodiments, M ¹, M², M3 at each occurrence,independently comprises a fused-multicyclic aryl moiety comprising atleast two fused rings.

In certain specific embodiments, M², M², or M³ are, at each occurrence,independently selected from the group consisting of adimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY,his-fluorophenyl-BODIPY, acridine, terrylene, sexiphenyl, porphyrin,benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl,(bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl,bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl,squaraine, squarylium, 9,10-ethynylanthracene, and ter-naphthyl moiety.

In some embodiments, M¹, M², or M³ are, at each occurrence,independently selected from the group consisting of p-terphenyl,perylene, azobenzene, phenazine, phenanthroline, acridine,thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide,perylene amide, and derivatives thereof. In some embodiments, M ¹, M²,M3 are, at each occurrence, independently selected from the groupconsisting of a coumarin dye, resorufin dye, dipyrrometheneborondifluoride dye, ruthenium bipyridyl dye, thiazole orange dye,polymethine, and N-aryl-1,8-naphthalimide dye. In certain embodiments, M¹, M², or M3 are, at each occurrence, independently selected from thegroup consisting of a coumarin dye, boron-dipyrromethene, rhodamine,cyanine, pyrene, perylene, perylene monoimide, 6-FAM, 5-FAM, 6-FITC,5-FITC, and derivatives thereof.

In some embodiments, M¹, M², or M³ at each occurrence, independentlyhave one of the following structures:

In some embodiments, M¹, M², or M³ at each occurrence, independentlyhave one of the following structures:

In some embodiments, each occurrence of L⁸ independently comprises oneor more fused, aryl, or heteroaryl ring systems. In certain embodiments,each occurrence of L⁸ independently comprises one or more, fused,bicyclic or tricyclic, aryl, or heteroaryl ring system. In certainembodiments, the one or more, fused, bicyclic or tricyclic, aryl, orheteroaryl ring system has one of the following structures:

wherein:

a¹, a² and a³ are, at each occurrence, independently a 5, 6 or7-membered carbocyclic or heterocyclic ring; and

L⁶ is a direct bond or a linker.

In certain specific embodiments, L⁸, at each occurrence, independentlyhas one of the following structures:

In some embodiments, each occurrence of L⁸ independently comprises aphosphodiester. In certain embodiments, at least one occurrence of L⁸comprises ethylene oxide. In more specific embodiments, at least oneoccurrence of L⁸ comprises one of the following structures:

wherein:

g is an integer ranging from 1-10; and

z′ is an integer ranging from 1-30.

In some of the foregoing embodiments, z′ is 3, 6, or 11-28. In someembodiments, g ranges from 2-5. In other more specific embodiments, atleast one occurrence of L⁸ comprises the following structure:

In certain embodiments, each occurrence of L⁸ comprises the followingstructure:

In some specific embodiments, the compound is a compound selected fromTable 2. In other specific embodiments, the compound is a compoundselected from Table 3. The compounds in Tables 2 and 3 were preparedaccording to the procedures set forth in the Examples and their identityconfirmed by mass spectrometry.

Nos. Structure I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

Structure

As used in Tables 2 and 3 above and throughout this disclosure, F and F′refer to a fluorescein moiety have the following structures,respectively:

As used in Table 2 above and throughout this disclosure, Bs refers to amoiety having the following structure:

As used in Table 2 above and throughout this disclosure, FITC refers toa moiety having the following structure:

As used in Table 2 above and throughout this disclosure, AF555 refers toa moiety having the following structure:

As used in Table 2 above and throughout this disclosure, Cy3 refers to amoiety having one of the following structures:

As used in Table 2 above and throughout this disclosure, AF700 refers tothe fluorescent dye, Alexa Fluor 700 having a CAS Registry No. of1246956-22-8.

As used in Table 2 above and throughout this disclosure, AF680 refers toa moiety having the following structure:

As used in Table 2 above and throughout this disclosure, AF647 refers toa moiety having the following structure:

As used in Table 2 above and throughout this disclosure, AF350 refers toa moiety having the following structure:

As used in Table 2 above and throughout this disclosure, PB refers to amoiety having the following structure:

As used in Table 2 above and throughout this disclosure, AF594 refers toa moiety having the following structure:

As used in Table 2 above and throughout this disclosure dT refers to thefollowing structure:

wherein:

R is H or a direct bond.

Alternatively, for any of the embodiments of dye moieties disclosedherein, the oxo moiety

may be replaced with a direct bond to the remainder of the molecule. Insome of the foregoing embodiments, L¹ is alkylene, e.g., methylene orethylene.

The presently disclosed dye combinations are “tunable,” meaning that byproper selection of the variables in any of the foregoing compounds, oneof skill in the art can arrive at a compound having a desired and/orpredetermined FRET properties (e.g., fluorescence emission signal,Stoke's shift). The “tunability” of the compounds allows the user toeasily arrive at compounds having the desired Stoke's shift,fluorescence signal and/or color for use in a particular assay or foridentifying a specific analyte of interest. Although all variables mayhave an effect on the FRET properties of the compounds, proper selectionof M¹, M², M³, L⁴, m, and n is believed to play an important role in themolar fluorescence of the compounds. Accordingly, in one embodiment isprovided a method for obtaining a compound having a desired FRETfluorescence properties, the method comprising selecting M¹, M², and M³moieties having known interactive properties, preparing a compound ofstructure (I), (II), (III), or (IV) comprising the M¹, M², and M³moieties, and selecting the appropriate variables for L⁴, m, and n toarrive at the desired FRET properties (e.g., Stoke's shift, reduction ofdonor emission signal).

FRET fluorescence emission signal in certain embodiments can beexpressed in terms of the fold increase or decrease relative to the FRETfluorescence emission signal of the parent fluorophore(s) (e.g.,monomers). In some embodiments the FRET fluorescence emission signal ofthe present compounds is 1.1×, 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×,10×, or greater than 10× relative to the parentchromophore(s)/fluorophore(s). Various embodiments include preparingcompounds having the desired fold increase in fluorescence relative tothe parent fluorophore by proper selection of L⁴, m, and n.

For ease of illustration, various compounds comprising phosphorousmoieties (e.g., phosphate and the like) are depicted in the anionicstate (e.g., —OPO(OH)O⁻, —OPO₃ ²⁺). One of skill in the art will readilyunderstand that the charge is dependent on pH and the uncharged (e.g.,protonated or salt, such as sodium or other cation) forms are alsoincluded in the scope of embodiments of the disclosure.

Compositions comprising any of the foregoing compounds and one or moreanalyte molecules (e.g., biomolecules) are provided in various otherembodiments. In some embodiments, use of such compositions in analyticalmethods for detection of the one or more analyte molecules is alsoprovided. In still other embodiments, the compounds are useful invarious analytical methods. For example, in certain embodiments thedisclosure provides a method of staining a sample, the method comprisingadding to said sample a compound of any of the foregoing embodiments(e.g., a compound of structure (I), (II), (III), or (IV)), in an amountsufficient to produce an optical response when said sample isilluminated at an appropriate wavelength.

In some embodiments of the foregoing methods, R² is a linker comprisinga covalent linkage to an analyte molecule, such as a biomolecule. Forexample, a nucleic acid or polymer thereof, or an amino acid or apolymer thereof (e.g., polynucleotide or polypeptide). In still moreembodiments, the biomolecule is an enzyme, receptor, receptor ligand,antibody, glycoprotein, aptamer, or prion.

In yet other embodiments of the foregoing method, R² is a linkercomprising a covalent linkage to a solid support such as a microparticle(e.g., controlled pore glass or polystyrene beads). For example, in someembodiments the microparticle is a polymeric bead or non-polymeric bead.

In even more embodiments, the optical response is a fluorescentresponse.

In other embodiments, said sample comprises cells, and some embodimentsfurther comprise observing said cells by flow cytometry.

In still more embodiments, the method further comprises distinguishingthe fluorescence response from that of a second fluorophore havingdetectably different optical properties.

In other embodiments, the disclosure provides a method for visuallydetecting an analyte molecule, such as a biomolecule, comprising:

(a) providing a polymer compound according to the foregoing embodiments(e.g., structure (I), (II), (III) or (IV)), wherein the polymer compoundcomprises covalent bond to the analyte molecule (e.g., one of R² or R³is a linker comprising a covalent bond to the analyte molecule, and theother of R² or R³ is H, OH, alkyl, alkoxy, alkylether or—OP(═R_(a))(R_(b))R_(c)); and

(b) detecting the compound by its visible properties.

In some embodiments the analyte molecule is a nucleic acid or polymerthereof, or an amino acid or a polymer thereof (e.g., polynucleotide orpolypeptide). In still more embodiments, the analyte molecule is anenzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer, orprion.

In other embodiments, a method for visually detecting an analytemolecule, such as a biomolecule is provided, the method comprising:

(a) admixing any of the foregoing polymer compounds wherein the compoundcomprises a covalent bond to Q selected from Table 1, with the analytemolecules; and

(b) forming a bio-conjugate of the polymer compound and the analytemolecule; and

(c) detecting the bio-conjugate by its visible properties.

Some embodiments provide use of the composition in an analytical methodfor detection of the one or more analyte molecules.

In addition to the above methods, embodiments of the compounds ofstructure (I), (II), (III) and (IV) find utility in various disciplinesand methods, including but not limited to: imaging in endoscopyprocedures for identification of cancerous and other tissues;single-cell and/or single molecule analytical methods, for exampledetection of polynucleotides with little or no amplification; cancerimaging, for example by conjugating a compound of structure (I), (II),(III) and (IV) to an antibody or sugar or other moiety thatpreferentially binds cancer cells; imaging in surgical procedures;binding of histones for identification of various diseases; drugdelivery, for example by replacing the M¹, M² and/or M³ moiety in acompound of structure (I), (II), (III) and (IV) with an active drugmoiety; and/or contrast agents in dental work and other procedures, forexample by preferential binding of the compound of structure (I), (II),(III) and (IV) to various flora and/or organisms.

It is understood that any embodiment of the compounds of structure (I),(II), (III) and (IV), as set forth above, and any specific choice setforth herein for a R¹, R², R³, R⁴, R⁵, L¹, L², L³, L⁴, L⁵, L⁶, L⁷,L^(7b), L⁸, M¹, M², M³, m, and/or n variable in the compounds ofstructures (I), (II), (III) or (IV), as set forth above, may beindependently combined with other embodiments and/or variables of thecompounds of structures (I), (II), (III) and (IV) to form embodiments ofthe disclosure not specifically set forth above. In addition, in theevent that a list of choices is listed for any particular R¹, R², R³,R⁴, R⁵, L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L^(7b), L⁸, M¹, M², M³, m, and/or nvariable in a particular embodiment and/or claim, it is understood thateach individual choice may be deleted from the particular embodimentand/or claim and that the remaining list of choices will be consideredto be within the scope of the disclosure.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in theprocess described herein the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

Furthermore, all compounds of the disclosure which exist in free base oracid form can be converted to their salts by treatment with theappropriate inorganic or organic base or acid by methods known to oneskilled in the art. Salts of the compounds of the disclosure can beconverted to their free base or acid form by standard techniques.

The following Reaction Schemes illustrate exemplary methods of makingcompounds of this disclosure. It is understood that one skilled in theart may be able to make these compounds by similar methods or bycombining other methods known to one skilled in the art. It is alsounderstood that one skilled in the art would be able to make, in asimilar manner as described below, other compounds of structure (I),(II), (III), or (IV) not specifically illustrated below by using theappropriate starting components and modifying the parameters of thesynthesis as needed. In general, starting components may be obtainedfrom sources such as Sigma Aldrich, Lancaster Synthesis, Inc.,Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this disclosure.

In particular, methods for preparing embodiments of the compoundsdisclosed herein (e.g., structures (I), (II), (III), and (IV)) can befound, for example, in PCT Pub. Nos. WO 2015/027176, WO 2016/138461, WO2016/183185, WO 2017/173348, WO 2017/173355, WO 2017/177065, WO2017/196954, WO 2017/214165, and WO 2018/022925, each of which arehereby incorporated by reference in their entirety.

Reaction Scheme I illustrates an exemplary method for preparing anintermediate useful for preparation of compounds of structures (I),(II), (III), and (IV), where R′, L², and L³ are as defined above, R² andR³ are as defined above or are protected variants thereof and L is anoptional linker. Referring to Reaction Scheme 1, compounds of structurea can be purchased or prepared by methods well-known to those ofordinary skill in the art. Reaction of a with M-X, where X is a halogensuch as bromo, under Suzuki coupling conditions known in the art resultsin compounds of structure b. Compounds of structure b can be used forpreparation of compounds of structures (I), (II), (III), or (IV) asdescribed below.

Reaction Scheme II illustrates an alternative method for preparation ofintermediates useful for preparation of compounds of structures (I),(II), (III), and (IV).

Referring to reaction Scheme II, where R¹, L¹, L², L³, and G as definedabove, and R² and R³ are as defined above or are protected variantsthereof, a compound of structure c, which can be purchased or preparedby well-known techniques, is reacted with M-G′ to yield compounds ofstructure d. Here, G and G′ represent functional groups havingcomplementary reactivity (i.e., functional groups which react to form acovalent bond). G′ may be pendant to M or a part of the structuralbackbone of M. G may be any number of functional groups describedherein, such as an alkyne or amine.

The compound of structures (I), (II), (III), and (IV) may be preparedfrom one of structures b or d by reaction under well-known automated DNAsynthesis conditions with a phosphoramidite compound having thefollowing structure (e):

wherein each X is independently a desired monomer unit (e.g., a dyecontaining moiety, a linker, etc.).

DNA synthesis methods are well-known in the art. Briefly, two alcoholgroups, for example R² and R³ in intermediates b or d above, arefunctionalized with a dimethoxytrityl (DMT) group and a2-cyanoethyl-N,N-diisopropylamino phosphoramidite group, respectively.The phosphoramidite group is coupled to an alcohol group, typically inthe presence of an activator such as tetrazole, followed by oxidation ofthe phosphorous atom with iodine. The dimethoxytrityl group can beremoved with acid (e.g., chloroacetic acid) to expose the free alcohol,which can be reacted with a phosphoramidite group. The 2-cyanoethylgroup can be removed after oligomerization by treatment with aqueousammonia.

Preparation of the phosphoramidites used in the oligomerization methodsis also well-known in the art. For example, a primary alcohol (e.g., R³)can be protected as a DMT group by reaction with DMT-Cl. A secondaryalcohol (e.g., R²) is then functionalized as a phosphoramidite byreaction with an appropriate reagent such as 2-cyanoethylN,N-dissopropylchlorophosphoramidite. Methods for preparation ofphosphoramidites and their oligomerization are well-known in the art anddescribed in more detail in the examples.

Compounds of structures (I), (II), (III), and (IV) are prepared byoligomerization of intermediates b or d and e according to thewell-known phophoramidite chemistry described above. The desired numberof m and n repeating units is incorporated into the molecule byrepeating the phosphoramidite coupling the desired number of times.

In various other embodiments, compounds useful for preparation of thecompound of structures (I), (II), (III), and (IV) are provided. Thecompounds can be prepared as described above in monomer, dimer and/oroligomeric form and then the M¹, M², and/or M³ moieties covalentlyattached to the compound via any number of synthetic methodologies(e.g., the “click” reactions described above) to form compounds ofstructures (I), (II), (III), and (IV).

The efficiency of the FRET process depends, in part, on characteristicsof the chromophores. Specifically, high efficiency FRET requires a largeoverlap between the absorbance spectrum of the donor chromophore and theemission spectrum of the acceptor chromophore (i.e., the J-value).Additionally, the distance and orientation of the chromophores plays animportant role. FRET efficiency is inversely proportional to the 6^(th)power of the distance between the chromophores and the angle of thetransition dipole moment should substantially align to be parallel orantiparallel (i.e., be near to 0° or)180° . Accordingly, in certainembodiments, covalent attachments of a first and a second chromophore tothe polymer backbone are selected so distance between the first andsecond chromophore is minimized and transition dipole momentssubstantially align. The efficiency of FRET can be expressed accordingto the following equation:

$E_{{FRET} =}\frac{Ro^{6}}{{Ro^{6}} + R^{6}}$

wherein EFRET is FRET efficiency, R is the distance betweenchromophores, and Ro is expressed according to the following equation:

Ro=(8.8×10²³ JK ² Q _(o) n ⁻⁴)^(1/6)

wherein J is the spectral overlap of the absorbance spectrum of theacceptor and the emission spectrum of the donor, Qo is donor quantumefficiency, n⁻⁴ is the index of medium between the donor and acceptor(constant), and K² is the dipole directions matching.

Accordingly, one embodiment provides a polymer compound comprising anacceptor chromophore having an acceptor transition dipole moment andbeing covalently linked to a polymer backbone and a donor chromophorehaving a donor transition dipole moment and being covalently linked tothe polymer backbone, wherein the acceptor chromophore and donorchromophore have a J-value greater than about 1 x 10¹⁰ and the polymercompound adopts a confirmation in solution at physiological conditionswherein the effective distance between the acceptor chromophore and thedonor chromophore is less than about 50.0 nm and the acceptor transitiondipole and the donor transition dipole are substantially parallel orsubstantially antiparallel.

In some embodiments, the acceptor chromophore and donor chromophore havea J-value greater than about 1×10¹¹. In certain embodiments, theacceptor chromophore and donor chromophore have a J-value greater thanabout 1×10¹². In some more specific embodiments, the acceptorchromophore and donor chromophore have a J-value greater than about1.2×10¹². In certain more specific embodiments, the acceptor chromophoreand donor chromophore have a J-value greater than about 1.5×10¹².

In some embodiments, the effective distance between the acceptorchromophore and the donor chromophore is less than about 25.0 nm. Incertain embodiments, the effective distance between the acceptorchromophore and the donor chromophore is less than about 10.0 nm.

In some embodiments, the acceptor chromophore is a fluorescent dyemoiety. In certain embodiments, the acceptor chromophore has an emissionmaxima ranging from about 480 nm to about 740 nm. In more specificembodiments, the acceptor chromophore has an emission maximum of about488 nm, about 500 nm, about 506 nm, about 519 nm, about 522 nm, about528 nm, about 564 nm, about 573, about 591 nm, about 603 nm, about 616nm, about 622 nm, about 640 nm, about 650 nm, about 666 nm, about 679nm, 714 nm, or about 731 nm.

In some embodiments, the acceptor chromophore has one of the followingstructures:

In some embodiments, the donor chromophore is a fluorescent dye moiety.In certain embodiments, the donor chromophore has an absorbance maximumat about 350 nm, at about 405 nm or at about 488 nm. In some morespecific embodiments, the donor chromophore has one of the followingstructures:

In some embodiments, the acceptor chromophore and the donor chromophoreare both fluorescent dye moieties.

In certain embodiments, the angle between the acceptor transition dipolemoment and the donor transition dipole moment ranges from 120° to 180° .In some more specific embodiments, the angle between the acceptortransition dipole moment and the donor transition dipole moment rangesfrom 0° to 60° . In certain more specific embodiments, the polymercompound further comprises a first acceptor chromophore is covalentlylinked at a proximal end of the polymer backbone, and a second acceptorchromophore is covalently linked at a distal end of the polymerbackbone; and donor chromophore is covalently linked between theproximal and distal ends of the polymer backbone.

In more specific embodiments, the first acceptor chromophore and thesecond acceptor chromophore are the same. In some embodiments, thepolymer backbone comprises a phosphate linker. In more embodiments, thepolymer backbone comprises an alkylene oxide linker. In someembodiments, the alkylene oxide is ethylene oxide.

In some embodiments, the polymer compound has a molecular weight lessthan 20,000 g/mol. In some embodiments, the polymer compound has amolecular weight less than 19,000 g/mol, 18,500 g/mol, 18,000 g/mol,17,500 g/mol, 17,000 g/mol, 16,500 g/mol, 16,000 g/mol, 15,500 g/mol,15,000 g/mol, 14,500 g/mol, 14,000 g/mol, 13,500 g/mol, 13,000 g/mol,12,500 g/mol, 11,500 g/mol, 11,000 g/mol, 10,500 g/mol, 10,000 g/mol,9,500 g/mol, 9,000 g/mol, 8,500 g/mol, 8,000 g/mol, 7,500 g/mol, 7,000g/mol, 6,500 g/mol, 6,000 g/mol, 5,500 g/mol, 5,000 g/mol, 4,500 g/mol,4,000 g/mol, 3,500 g/mol, 3,000 g/mol, 2,500 g/mol, 2,000 g/mol, 1,500g/mol, or 1,000 g/mol.

In some embodiments the polymer compound is not a peptide or protein. Insome other embodiments, the polymer backbone has no amide bonds.

The following Examples are provided for purposes of illustration, notlimitation.

EXAMPLES General Methods

Mass spectral analysis was performed on a Waters/Micromass Quattro microMS/MS system (in MS only mode) using MassLynx 4.1 acquisition software.Mobile phase used for LC/MS on dyes was 100 mM1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH8. Phosphoramidites and precursor molecules were also analyzed using aWaters Acquity UHPLC system with a 2.1mm×50mm Acquity BEH-C18 columnheld at 45° C., employing an acetonitrile/water mobile phase gradient.Molecular weights for monomer intermediates were obtained usingtropylium cation infusion enhanced ionization on a Waters/MicromassQuattro micro MS/MS system (in MS only mode). Excitation and emissionprofiles experiments were recorded on a Cary Eclipse spectra photometer.

All reactions were carried out in oven dried glassware under a nitrogenatmosphere unless otherwise stated. Commercially available DNA synthesisreagents were purchased from Glen Research (Sterling, Va.). Anhydrouspyridine, toluene, dichloromethane, diisopropylethyl amine,triethylamine, acetic acid, pyridine, and THF were purchased fromAldrich. All other chemicals were purchased from Aldrich or TCI and wereused as is with no additional purification.

Example 1 Synthesis of Polymer Backbone and Derivatization Synthesis ofRepresentative Polymer Compounds

Polymers are synthesized using an Applied Biosystems 394 DNA/RNAsynthesizer on a 0.5-1 μmol scale. The polymer compounds are synthesizeddirectly on

CPG beads or a polystyrene solid support. Synthesis is carried out inthe 3′ to 5′ direction using standard solid phase DNA syntheticmethodology (i.e., β-cyanoethyl phosphoramidite coupling chemistry).Monomer reagents (e.g., Fluoroside phosphoramidites, C₂ alkylphosphoramidite, hexaethylene glycol phosphoramidite, aminophosphoramidites, and alkyne phosphoramidites) are dissolved inacetonitrile and dichloromethane to make a 0.1 M stock solution. Monomerreagents are added in successive order using the following synthesiscycle:

1) removal of the 5′-dimethoxytrityl protecting group withdichloroacetic acid in dichloromethane or toluene;

2) coupling the next phosphoramidite monomer reagent with activatorreagent (0.2 M ETT) in acetonitrile;

3) oxidation of phosphate (III) to form stable phosphate (V) withiodine/pyridine/water; and

4) capping unreacted 5′-hydroxyl groups with aceticanhydride/1-methylimidizole/acetonitrile.

The synthesis cycle is repeated until the desired polymer compound isassembled (e.g., compounds of Table 2 or 3). Upon completion, theterminal monomethoxytrityl (MMT) group or dimethoxytrityl (DMT) group isremoved using dichloroacetic acid in dichloromethane or toluene.Following synthesis, the support is treated with 20% diethylamine inacetonitrile for 15 minutes.

The polymers are then cleaved from the solid support using concentratedaqueous ammonium hydroxide at 55° C. for 2 hours. Constructs arecharacterized by ESI-MS to confirm mass and purity. Concentration isdetermined by UV.

Chromophore Coupling to Amine/Alkyne Polymer Compounds

Representative polymer constructs are synthesized to have orthogonalreactive groups (e.g., amine and alkyne) so distinct dyes moieties couldbe added. For instance, click chemistry was used to attach a firstfluorophore to alkynes of the polymer and in a later synthetic step asecond fluorophore is attached to amines of the polymer. Alternatively,fluorophores can be added during the DNA synthetic step by inclusion ofa fluorophore containing phosphoramidite during step 2 described above.

For the post synthetic modifications, a 100 mM sodium ascorbate solutionand 500 mM phosphate buffer pH 7.6 are prepared. A first desired dyemoiety comprising an azide functional group is dissolved in dimethylsulfoxide at a concentration of 100 mM (“dye solution”).

The 100 mM sodium ascorbate solution (i.e., a 2.5:1:1 mixture of 100 mMsodium ascorbate/100 mM THTPA/50 mM copper sulfate) is prepared byadding equal volumes of THTPA and copper sulfate and allowing themixture to sit for 5-10 minutes followed by the addition of sodiumascorbate.

The phosphate buffer, dye solution, polymer compound, and extra DMSO aremixed together followed by the sodium ascorbate solution. Reactions areprepared having final polymer concentration of 0.5 mM, phosphate bufferconcentration of 100 mM, 50-60% DMSO and 5-10 fold excess dye. Reactionsare placed in the dark overnight on a rotator or vortex. Samples arediluted with water and desalted using G-25 Sephadex resin. Samples arecollected and characterized by ESI-MS to confirm mass and purity.Following sample analysis the polymer compounds are lyophilized.

Addition of a second desired dye moiety (e.g., fluorophore or otherlabel) to representative polymer compounds is achieved usingNHS-ester/amine coupling chemistry. A sodium tetraborate buffer pH 9stock solution (“borate buffer”) is initially prepared. A second dyemoiety comprising an NETS-ester is dissolved at a concentration of100-200 mM in DMSO. Reactions are prepared to have a polymerconcentration of 1-2 mM, a borate buffer concentration of 100 mM, 30-75%DMSO, and a 5-10-fold excess of dye. Reactions are placed in the darkovernight on a rotator or vortex. Samples are diluted with water anddesalted using G-25 Sephadex resin. Samples are collected andcharacterized by ESI-MS to confirm mass and purity. Additionally,characterization by UV and fluorescence spectroscopy is used todetermine FRET properties.

Example 2 Compound Emission Characterization

Representative polymer compounds of structure (III) (compounds I-1, I-3,1-5 and 1-7) and structure (IV) (compounds 1-2, 1-4, 1-6 and 1-8) wereprepared according to the methods described herein in Example 1.Compounds of structure (III) where synthesized wherein the M² moiety,being a donor chromophore, was Cy3. Compounds of structure (IV) wheresynthesized wherein the M³ moiety, being a donor chromophore, was AF555.It is well established in the art that Cy3 and AF555 are spectrallyequivalent as isolated species, and this study allows for preliminaryevaluation of spectroscopic differences in more complex environments.Chromophores for each of the two M¹ moieties in each compound wereselected from AF680, AF647, AF594 and AF700 and were the same in eachcompound.

Test compounds were prepared in solution at concentrations between 1 and10 μM and a pH of 7.0. Samples were tested using an excitationwavelength of 488 nm and the resultant spectra are shown in FIGS. 2through 9 . As the data show, the emission signals from the acceptorchromophores are substantially similar, however certain embodimentsdemonstrate better energy transfer from the donor to acceptorchromophores as evidenced by lessened relative fluorescence from thedonor fluorophore, see for example, the smaller emission peaks in FIG. 4(better energy transfer) relative to the equivalent peak in FIG. 5 .

FIG. 10 provides a positional overly of the emission spectra for thecompounds of structure (III) and demonstrates the range of Stokes shiftobserved for the different embodiments of the invention. As determinedfrom the emission maximum relative to the excitation wavelength, theStokes shift for compounds I-7, I-1, I-3 and I-5 were 243 nm, 226 nm,191 nm and 134 nm, respectively.

Example 3 Demonstration of Efficient FRET Emission

To demonstrate high efficiency emission due to FRET for embodiments ofthe invention, the emission spectra of Compound I-10, containing donorand acceptor chromophores was compared to a related compound devoid of adonor chromophore, but containing the same type and number of acceptorchromophores.

Compound I-10 was prepared according to the methods described herein inExample 1 and was synthesized having the donor chromophore AF350. Theacceptor chromophore present on both ends of the molecule was thefluorescein derivative, FITC.

The di-fluorescein comparison compound, Compound A, which is devoid of adonor chromophore is provided below:

wherein F is the fluorescein moiety:

Test compounds were prepared in solution at a concentration 350 nM and apH of 7.0. Samples were evaluated using an excitation wavelength of 350nm and the resulting overlaid spectra are shown in FIG. 11 . Theobserved Stokes shift for the emission maximum of 520 nm for CompoundI-10 is 170 nM.

Compound I-10 showed a ˜5.5 fold increase in fluorescence relative toCompound A demonstrating the effective transfer of excitation energyfrom the donor chromphor (AF350) to the acceptor, FITC moieties toprovide the observed increase.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, including U.S. Patent Application No.62/906,591, filed Sep. 26, 2019, are incorporated herein by reference,in their entirety to the extent not inconsistent with the presentdescription.

From the foregoing it will be appreciated that, although specificembodiments of the disclosure have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the disclosure. Accordingly, the disclosure isnot limited except as by the appended claims.

1. A compound having the following structure (I):

or a stereoisomer, salt, or tautomer thereof, wherein: M¹ and M² are, ateach occurrence, independently a chromophore, provided that at least oneof M¹ and M² is a FRET donor, and another one of M¹ and M² is acorresponding FRET acceptor; L¹ is, at each occurrence, an optionallinker, provided that at least one occurrence of L¹ is present andcomprises oxygen; L² and L³ are, at each occurrence, independently anoptional alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, heteroalkynylene or heteroatomic linker; L⁴ is, ateach occurrence, independently an alkylene or heteroalkylene linker; R¹is, at each occurrence, independently H, alkyl or alkoxy; R² and R³ areeach independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl,—OP(═R_(a))(R_(b))R_(c), Q, or a protected form thereof, or L′; R⁴ is,at each occurrence, independently OH, SH, O⁻, S⁻, OR_(d) or SR_(d); R⁵is, at each occurrence, independently oxo, thioxo or absent; R_(a) is Oor S; R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d); R_(c) is OH, SH, O⁻,S⁻, OR_(d), OL′, SR_(d), alkyl, alkoxy, heteroalkyl, heteroalkoxy,alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether; R_(d) is acounter ion; Q is, at each occurrence, independently a moiety comprisinga reactive group, or protected form thereof, capable of forming acovalent bond with an analyte molecule, a targeting moiety, a solidsupport, or a complementary reactive group Q′; L¹ is, at eachoccurrence, independently a linker comprising a covalent bond to Q, alinker comprising a covalent bond to a targeting moiety, a linkercomprising a covalent bond to an analyte molecule, a linker comprising acovalent bond to a solid support, a linker comprising a covalent bond toa solid support residue, a linker comprising a covalent bond to anucleoside or a linker comprising a covalent bond to a further compoundof structure (I); m is, at each occurrence, independently an integer ofzero or greater; and n is an integer of one or greater. 2-9. (canceled)10. The compound of claim 1, wherein the compound has the followingstructure (IA):

wherein: z is, at each occurrence, independently an integer from 1 to100; and m is, at each occurrence, independently an integer from 0 to 6.11-13. (canceled)
 14. The compound of claim 10, wherein z is, at eachoccurrence, independently an integer from 1 to
 30. 15. (canceled) 16.The compound of claim 10, wherein m is, at each occurrence,independently an integer from 2-4.
 17. (canceled)
 18. The compound ofclaim 10, wherein the compound has the following structure (TB):

wherein: x¹, x², x³, and x⁴ are, at each occurrence, independently aninteger from 0 to
 6. 19-28. (canceled)
 29. The compound of claim 1,wherein at least one occurrence of L¹ is a linker comprising a triazolylfunctional group.
 30. The compound of claim 29, wherein at least oneoccurrence of L¹-M¹ or L¹-M² comprises one of the following structures:

wherein L^(a) and L^(b) are each independently optional linkers. 31-33.(canceled)
 34. The compound of claim 30, wherein L^(a) or L^(b), orboth, is absent.
 35. The compound of claim 30, wherein L^(a) or L^(b),or both, is present.
 36. The compound of claim 35, wherein L^(a) andL^(b), when present, are each independently alkylene or heteroalkylene.37. The compound of claim 35, wherein L^(a) and L^(b) independently haveone of the following structures:


38. (canceled)
 39. The compound of claim 1, wherein L¹ comprises one ofthe following structures:

wherein a, b, and c are each independently an integer ranging from 1-6.40-46. (canceled)
 47. The compound of claim 1, wherein R⁴ is, at eachoccurrence, independently OH, —O or OR_(d); R⁵ is, at each occurrence,oxo; and R¹ is, at each occurrence, H.
 48. (canceled)
 49. (canceled) 50.The compound of claim 1, wherein one of R² or R³ is OH or—OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is Q or a linkercomprising a covalent bond to Q. 51-60. (canceled)
 61. The compound ofclaim 1, wherein Q has one of the following structures:

wherein each X is independently a halogen.
 62. (canceled)
 63. Thecompound of claim 1, wherein one of R² or R³ is OH or—OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is a linkercomprising a covalent bond to an analyte molecule or a linker comprisinga covalent bond to a solid support. 64-66. (canceled)
 67. The compoundof claim 1, wherein R² or R³ has one of the following structures:

68-76. (canceled)
 77. The compound of claim 1, wherein at least onecombination of M¹ and M² is a FRET pair with a J-value greater thanabout 1×10¹⁰. 78-81. (canceled)
 82. The compound of claim 1, whereinM¹and M², are, at each occurrence, independently selected from the groupconsisting of a dimethylaminostilbene, quinacridone,fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine,terrylene, sexiphenyl, porphyrin, benzopyrene,(fluorophenyl-dimethyl-difluorobora-dimethyl-difluorobora-diaza-indacene)phenyl,(bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl,bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl,squaraine, squarylium, 9,10-ethynylanthracene, and ter-naphthyl moiety;ii) p-terphenyl, perylene, azobenzene, phenazine, phenanthroline,acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, peryleneimide, perylene amide, and derivatives thereof; iii) a coumarin dye,resorufin dye, dipyrrometheneboron difluoride dye, ruthenium bipyridyldye, thiazole orange dye, polymethine, and N-aryl-1,8-naphthalimide dye;or iv) a coumarin dye, boron-dipyrromethene, rhodamine, cyanine, pyrene,perylene, perylene monoimide, 6-FAM, 5-FAM, 6-FITC, 5-FITC, andderivatives thereof. 83-85. (canceled)
 86. The compound of claim 1,wherein M¹ and M², at each occurrence, independently have one of thefollowing structures:

87-98. (canceled)
 99. The compound of claim 1, wherein the compound isselected from:

wherein: Bs has the following structure:

FITC has the following structure:

AF555 has the following structure:

Cy3 has the following structure:

AF700 refers to Alexa Fluor 700 having a CAS Registry No. of1246956-22-8; AF680 has the following structure:

AF647 has the following structure:

AF350 has the following structure:

PB has the following structure:

AF594 has the following structure:

and dT has the following structure:

wherein: R is H or a direct bond. 100-129. (canceled)
 130. A compositioncomprising the compound of any one of claim 1 and one or more analytemolecules.
 131. (canceled)